Changes in polygalacturonase and α-amylase activities in melon fruit-香川大学学術情報リポジトリ

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Tech Bull Fac Agr Kagawa Univ, Vol 44, No 2, 175-179, 1992

CHANGES IN POLYGALACTURONASE AND a-AMYLASE

ACTIVITIES IN MELON FRUIT

Toshiyuki MATSUI and Y uichi YOSHIDA

This paper reported the changes in total ('TP) and water soluble pectin (WSP), and starch contents in three melon cultivars (Cucumts melo L ) in relation to polygalacturonase (PG) and a-amylase activities The properties of these enzymes in 'Ginsen' were also determined The PG activity in the water soluble fraction increased whereas that in the insoluble fraction decreased However, there were no significant differences in the T P and WSP contents between each harvest period T h e PG and a-amylase activities in both fractions, T P , WSP and starch contents cannnot be used a s indices of optimum harvest period in place of firmness

T h e PG and a-amylase of 'Ginsen' had a molecular weight of ca 24,000, and 52,000, optimum pH of ca 5 5 and 5 0, and km value of 0 14 and 3 3 mg/ml for polygalacturonic acid and starch, respectively Both enzymes had an optimum temperature of 45°C

Key Words : total pectin, water soluble pectin, polygalacturonase, a-amylase, melon

Introduction

Melon fruit is known to soften with ethylene treatment after harvest due to an enhancement of some degradative enzymes(12) PG is found to decompose polygalacturonic acid and polygalacturonan into galacturonic acid and its d e r i ~ a t i v e ' ~ ) whereas a-amylase degrades starch into maltose and its derivative T h e soluble solids content and firmness were used a s maturity indices of melon This study was therefore conducted to determine the changes in PG and a-amylase activities in relation to T P , W S P and starch contents of melons a t each indicated suitable harvest date after anthesis

Materials and Methods

Materials. The growing conditions were the same as in the previous paper(4) T h e fruits

were harvested at 5- 10 days interval

Enzyme extraction (PC and a-amylase). PG and a-amylase were extracted from about

10 g of fruit pulp, according to the methods of MA'TSUI and KITAGAWA("~)

Enzyme assay. The assay media for PG and a-amylase were prepared a s in the previous

Partial purification of PC and a-amylase. 'Ginsen' harvested at 20 days after anthesis

was used for partial purification of PG and a-amylase as described in the previous paper("2)

Estimation of the molecular weight of the enzyme by HPLC. The estimation procedure

was carried out as described in the previous paper(5) T h e elution volumes of the following molecular markers; cytochrome c (indicator protein, mol wt 1 25 x lo4 ), albumin (bovine, mol wt 6 7 x lo4 ) and aldolase (rabbit muscle, mol wt 1 6 x lo5 ) were plotted against the

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176 T e c h Bull F a c A g r Kagawa U n i v , Vol 44, No 2, 1992 molecular weight of the marker

Determination of total pectin ( TP ) and water soluble pectin ( WSP ). T P and W S P were analyzed according to the methods described in the previous paper('22)

Determination of starch. Starch content was analyzed by the same method a s in the

previous paper(2)

Measurement of Firmness. The fruit was cut longitudinally into two near the center T h e

firmness of pericarp at six points was measured using a penetrometer with a 5 mm conical plunger (Kiya Seisaku Co Ltd)

Results and Discussion

Proper ties o f polygalacturonase and a - amylase in melon fruits

I ) Optimum pH and temperature. Fig 1 shows the optimum pH (A) and temperature (B)

for the activities of partially purified PG and a-amylase The optimum pH for PG and a-amylase of 'Ginsen' was ca 5 5 and ca 5 0, respectively, and their optimum temperature was ca 45°C T h e optimum pH of PG in 'Ginsen' was the same as that of kiwifruit while the optimum temperature was slightly lower On the other hand, the optimum pH and temperature of a-amylase in 'Ginsen' were slightly lower than that of kiwifruit

2 ) Km value for galacturonic acid and soluble starch. The LINEWEAVER-BURK reciprocal

loo 80 ,-, 40

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Fig 1 Optimum pH ( A ) and temperature ( B ) for the Fig 2 Molecular weight of polygalacturonase and a - activites of polygalacturonase and a -amylase in amylase a s determined by HPLC (Shim-pack melon c v Ginsen , polygalacturonase; o, a - Diol-150) Molecular weight makers; A, aldolase amylase, (1 6 X lo5); B, albumin (6 7 x lo4); C, cytochrome ( A ) The activity at pH 5 0 ( a -amylase) and c (1 25X lo4) e and d indicate the molecular

pH 5 5 (PG) was taken a s 100% weight of polygalacturonase and a -amylase in ( B ) T h e activity at 45C was taken a s 100% melon cv Ginsen, respectively

Values are means of duplicate assays al 2 I

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T MAISUI and Y YOSHIDA : PG activity in melon 177 plot for PG and a-amylase as a function of polygalacturonic acid and soluble starch concentrations were linear and the km value was 0 14 mg/ml at pH 5 0 and 4 5 C , and 3 3 mg/ ml at pH 7 0 and 30°C, respectively

3) Molecular weight o f PG and a-amylase T h e molecular weight of PG and a-amylase

in 'Ginsen' was estimated as ca 24,000 and 52,000, respectively by Shim-pack Diol (Fig 2)

Changes in PG and a-amylase activities during growth of fruit. The suitable time to

harvest 'Honey Dew', 'IW-57' and 'Ginsen' was reported(4) to be about 50, 45 and 35 days after anthesis, respectively T h e average weight of these cultivars at harvest was 1,400, 750 and 490 g, respectively T h e PG activity in the water soluble fraction increased during maturity but this cannot be used a s a maturity index There were no significant differences in the PG activity in both the soluble and insoluble fractions between each harvest time@) (Table 1) On the other hand, a-amylase activity in both fractions was almost zero

Changes in T P , WSP and starch contents, and firmness during growth of fruit. The TP content in melon fruit was higher than W S P in all harvest period In 'IW-57', the TP was significantly higher than the WSP content at 40 days after anthesis No significant differences in. other cultivars were observed between 35 and 45 days after anthesis (Table 2) T h e highest T P content did not coincide with the period of highest PG activity These indicate that PG activity is not closely related to softening of the fruits and TP and W S P contents are not suitable chemical indices of the harvest maturity T h e starch content in melon appeared not to influence greatly the quality of the fruit since the increase in sucrose was less than 27 mg/100 g if the starch is completely converted to sucrose(r) (Fig 3) On the last harvest date, 'IW-57' was the most firm followed by 'Honey Dew' and 'Ginsen' (Fig 3) The suitable firmness of fruit for consumption however, was changed when melon was treated with ethylene after h a r v e d 4 )

Table 1 Changes in PG activity in the water soluble and insoluble fractions of three melon cultivars harvested at different days after anthesis*

Cultivars Galacturonate (L( mol/min/mg protein)/Days after. anthesis

10 20 30 35 40 45

Soluble fraction

'Honey Dew' 0 0 3 f 0 0 6 C 3 3 0 k 0 5 0 a b 400+006a 3 8 7 f 0 18ab 2 4 7 f 0 3 z a b 'IW-57' 0 0 3 k 0 0 5 ~ 1 5 1 f 0 5 6 b 1 1 9 f 0 0 4 ~ 2 6 0 f 0 0 1 a b 4 2 2 3 ~ 0 6 4 ~ 1 9 9 f 0 3 3 a b 'Ginsen' 0 6 0 f 0 33b 3 4 9 f 0 60a 2 7 8 f 0 27ab 2 9 4 f 0 0 8 ~ ~ 3 10+0 51ab

Insoluble fraction

'Honey Dew' 1 2 0 3 ~ 0 4 6 ~ 1 4 1 f 0 2 3 a 030+003a 0 2 9 f 0 03a 0 2 0 f 0 Ola 'IW-57' 0 31 f 0 loa 0 1 0 f 0 Ola 1 51 + 0 56a 0 3 0 f 0 03a 0 3 0 f 0 02a 0 20+0 Ola 'Ginsen' 1 4 9 f 0 0 2 a 0 . 3 5 + 0 0 5 ~ 0 0 5 f 0 0 2 ~ 0 0 5 f 0 0 1 b 0 5 0 + 0 0 6 ~

Average of three replications f standard error

* ~ n y two means having a common letter in the same cultivar are not significantly different at 5% level,

DMRT

6,

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178 Tech Bull Fac Agr Kagawa Univ , Vol 44, No 2, 1992 Table 2 Changes in total and water soluble pectin contents of three melon cultivars

harvested a t different days after anthesis*

Cultivars Days after anthesis

10 20 30 35 40 45

Total pectin content (rng/lOOg fresh wt.)

'Honey Dew' 421 f 53a 3 0 0 f ~8~ 2 4 9 f ~ 7 ~ 1 9 5 f gb ~ 3 3 f 2 8 ~ 'Iw-57" 887f213a 5 0 1 f l l c 554f 5bC 402f4' 854f2eab 401f GC 'Ginsen' 3 0 3 f 5Ta 3 1 5 f 13a 292+10a 2 7 4 f ga 251 f Za

Water soluble pectin content (mg/100g fresh wt.)

'Honey Dew' 2 5 0 f Za 2 0 1 + . 2 ~ 1 9 0 f 2b 1 8 6 + . 6 ~ 1 9 0 f 6b 'IW-57' 252f 2zabc 2 6 1 f

zabc

299, Za 2 8 3 f 4ab 5 0 f 3d 2 0 0 f 3' 'Ginsen' 8 5 f 13e 1 7 0 f 4bc 2 4 9 f l l a 2 0 0 f l l a b 1 6 9 f 8bc

Average of three replications f standard error

* ~ two means having a common letter in the same cultivar are not significantly different a t n ~ 5% level, DMRT

'Honey Dew 'Ginsen'

Days after anthesis Days after anthesis Fig3 Changes in starch content (A) and firmness ( B ) during

growth of melon fruit (A) Values are means with S E (n=3)

( B ) Values are means with S E (n=6)

Acknowledgement

W e wish to thank Miss KOBATA of Dimaru Food Co L t d for technical assistance

References (1) MATSUI, T and KITAGAWA, H : Seasonal

changes in pectinmethylesterase and polygalac- turonase activities in kiwifruit : Ntppon Shoku- htn Kogyo Gakkatsht, 35, 851 -855 (1988) (2) MATSUI, T and KIIAGAWA, H : Seasonal

changes in a-and ,8-amylase activities in relation to starch content in kiwifruit Ntppon

Shokuhtn Kogyo Gakkatsht, 36, 334-338

(1989)

(3) MATSUI, T and KITAGAWA, H : Effects of ethylene absorbent on polygalacturonase activity of persimmon fruit, J Japan Soc Hort Sct 57, 697-701 (1989)

(4) YOSHIDA, Y , OHI, M and FUJIMOTO, K :

Diferences in ripening behavior in some melon cultivars, J Japan Soc Hort Sct 58, 999- 1006 (1990)

(5)

T MKTSUI and Y YOSHIDA : PG activity in melon 179

(5) MATSUI, T , YOSHIDA, Y and KITAGAWA, H : Wiley and Sons, Inc, New York) pp 207-215 Changes in invertase activity of melon fruit in (1 984)

relation to sugar content, Ntppon Shokuhtn (7) AGRAVANTE, J U , MATSUI, T and KI~AGAWA,

Kogyo Gakkatsht 38, 871 -873 (1991) H : Effect of ethanol on ripening and amylase

(6) GOMEZ, K and GOMEZ, A A ; Statistical activity of banana, Ntppon Shokuhtn Kogyo procedure for Agricultural Research, (John Gakkatsht, 37, 235-238 (1990)

(Received June 30, 1992)

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